Methods for selecting enzymes having lipase activity

Abstract

Provided herein are methods and means for enhancing lipase activity. The system makes use of an emulsion for in vitro compartmentalization of a library of synthetic compounds which have a polynucleotide linked to a lipase substrate (e.g., a triglyceride). Expressed polypeptides having greater lipase activity will preferentially hydrolyze the substrate from the linked polynucleotide. Genes encoding polypeptides having less lipase activity will remain linked to the substrate and may be removed to enrich the library for more active variants. Also described are synthetic compounds and emulsions which can be used in the methods.

1 . A method of selecting for a polypeptide having lipase activity, the method comprising:
 (i) suspending a plurality of synthetic compounds in an aqueous phase, wherein the synthetic compounds individually comprise:
 (a) a polynucleotide encoding for a polypeptide, and 
 (b) a lipase substrate linked to said polynucleotide; and 
   wherein the aqueous phase comprises components for expression of the polypeptide;   (ii) forming a water-in-oil emulsion with the aqueous phase, wherein the synthetic compounds are compartmentalized in aqueous droplets of the emulsion;   (iii) expressing the polypeptides within the aqueous droplets of the emulsion, wherein a polypeptide with lipase activity in an aqueous droplet hydrolyzes one or more synthetic compounds in that droplet; and   (iv) separating the synthetic compounds to recover hydrolyzed and/or non-hydrolyzed synthetic compounds.   
     
     
         2 . The method of  claim 1 , wherein the lipase substrate is a triglyceride. 
     
     
         3 . The method of  claim 2 , wherein the polynucleotide encoding for a polypeptide is linked at the 2 position of the triglyceride. 
     
     
         4 . The method of  claim 1 , wherein the synthetic compounds further comprise a selectable marker. 
     
     
         5 . The method of  claim 4 , wherein the selectable marker is linked to the lipase substrate. 
     
     
         6 . The method of  claim 4 , wherein an expressed polypeptide having lipase activity in an aqueous droplet cleaves the selectable marker from one or more of the synthetic compounds in that droplet, thereby allowing selective removal of the non-hydrolyzed synthetic compounds of step (iv). 
     
     
         7 . The method of  claim 4 , wherein the selectable marker is linked to the polynucleotide encoding for a polypeptide. 
     
     
         8 . The method of  claim 4 , wherein the selectable marker is sequestered by the non-hydrolyzed synthetic compounds, thereby allowing selective removal of the hydrolyzed synthetic compounds of step (iv). 
     
     
         9 . The method of  claim 4 , wherein the selectable marker is an affinity tag. 
     
     
         10 . The method of  claim 9 , wherein the hydrolyzed synthetic compounds of step (iv) are separated from the non-hydrolyzed synthetic compounds with streptavidin. 
     
     
         11 . A synthetic compound comprising:
 (a) a polynucleotide encoding for a polypeptide; and   (b) a lipase substrate linked to said polynucleotide.   
     
     
         12 . The synthetic compound of  claim 11 , wherein the lipase substrate is a triglyceride. 
     
     
         13 . The synthetic compound of  claim 12 , wherein the polynucleotide encoding for a polypeptide is linked at the 2 position of the triglyceride. 
     
     
         14 . The synthetic compound of  claim 11 , wherein the polypeptide has lipase activity. 
     
     
         15 . The synthetic compound of  claim 11 , further comprising a selectable marker. 
     
     
         16 . The synthetic compound of  claim 15 , wherein the selectable marker is linked to the lipase substrate. 
     
     
         17 . The synthetic compound of  claim 15 , wherein the selectable marker is linked to the polynucleotide encoding for a polypeptide. 
     
     
         18 . The synthetic compound of  claim 11 , wherein the selectable marker is an affinity tag. 
     
     
         19 . A method of making the synthetic compound of  claim 11 , comprising:
 (i) linking a lipase substrate to a polynucleotide encoding for a polypeptide; and   (ii) recovering the synthetic compound.   
     
     
         20 . A polynucleotide library comprising a plurality of different synthetic compounds according to  claim 11 . 
     
     
         21 . A water-in-oil emulsion comprising the polynucleotide library of  claim 20 , wherein the synthetic compounds are compartmentalized in aqueous droplets of the emulsion. 
     
     
         22 . A method of making the emulsion of  claim 21 , comprising:
 (i) suspending the plurality of synthetic compounds in the aqueous phase; and   (ii) mixing the suspension of (i) with an oil.